Two-photon microscopy is a widely used, key method for functional imaging of cellular activity in living animals. Most recently, in vivo calciu imaging experiments have started to reveal the spatiotemporal activity patterns that occur in various areas of the neocortex during head-fixed mouse behavior. Typically, however, the field-of- view for imaging cellular activity is fairly small, on the order of a few hundred micrometers. This restriction limits the size of neuronal networks that can be studied and thus leaves open the question how local neuronal networks communicate with distant, synaptically connected regions. What is therefore needed is a new imaging instrument that allows high-resolution functional imaging from two regions simultaneously, in the best case identifying the mutual projection neurons. We have developed a novel multi-area 2-photon microscope (MA2PM) that fulfills this need by enabling simultaneous imaging of two sub-areas within a large global scan- field (1.8 mm diameter, a distance equivalent to lining 100,000 neuronal cell bodies side-by-side assuming 20 um per neuron). Such two areas can be independently and flexibly positioned, making it for example possible to simultaneously measure neurons in the primary and secondary somatosensory areas of mouse neocortex (> 1 mm apart) while the mouse is performing a tactile texture discrimination task with its whiskers. The Using the MA2PM prototype we have conducted first proof-of-principle experiments in somatosensory cortex of awake, behaving mice using a genetically-encoded calcium indicator. We apply viral retrograde labeling strategies to identify the subsets of neurons that give rise to the inter-areal connection. Our goal in the proposed project is to further optimize and extend this innovative, transforming microscopy technology in several ways. Aim 1: We will work on finalizing a full microscope design for 2-area imaging and demonstrate its usefulness for research on corticocortical processing. We are fully dedicated to build a modular, carefully designed instrument, perform a quantitative system characterization, and disseminate this new instrument to the broader neuroscience community, especially to the growing number of research groups applying two-photon imaging for the analysis of cortical processing. Aim 2: We will furthermore expand the system to an even larger field-of-view and extend it for simultaneous imaging of four sub-areas. To this end we will apply newest laser technology and employ state-of-the-art red-shifted genetically encoded calcium indicator for deep imaging. We particularly aim at instantiating two-layer imaging (e.g. in layers L2/3 and L5) in two connected cortical areas. Overall, we expect that the new multi-area imaging technology will be an enabling technology to bridge the level of local microcircuits to the 'macrocircuit' level of communicating brain areas and thus will be of immediate and broad interest and highest significance to the neuroscience community.